Understanding gbr-22 protein expression, purification and characterization
Introduction:
Proteins that are created from recombinant DNA from molecular cloning are known as recombinant proteins. These proteins are "used throughout biological and biomedical science" in areas such as x-ray crystallography and enzyme inhibition assays [1] . Some hosts for expressing the protein include bacteria, yeast, insect cells and human cells [1]. E. coli bacteria has become a popular expression host since many varieties of proteins can be over expressed in methods that "increase the yield of soluble protein" [1]. BL21 (DE3) is a common strain of E.coli that is used to quickly obtain large quantities of a protein. The plasmid that will be inserted into the expression host also contains some antibiotic-selection marker (e.g. ampicllin, carbenicillin) to identify bacterial colonies that successfully transformed with the plasmid [1]. After bacterial lysis through high-pressure homogenization or freeze-thaw procedures with lysozyme, the protein of interest is separated from extraneous proteins in a process known as protein purification [1]. To assist with protein purification, the modified recombinant protein includes an affinity tag, such as a C-terminal hexahistidine tag. The hexahistidine tag will bind to cations on a column matrix, and those proteins lacking the tag can be disposed of. Finally, the purified protein is characterized to ensure the protein is of adequate quality. SDS-PAGE analysis will denature the protein and show the quantity of the protein in proportion to the distance of migration in the gel. "This allows the purity of the sample to be estimated and whether the purified protein is of the expected size" [1]. UV-Vis spectroscopy is also used in this step to quantify the concentration of the protein. In this lab, the protein gbr22 was expressed, purified and characterized. As we complete the protocols in this lab, we expect the protein characterization to quantify the molecular weight of gbr22 near 25kDa, the literature value.
Materials & Methods:
Competent bacterial cells (New England BioLabs, E. Coli BL21(DE3)) were transformed with an expression plasmid pGEM-gbr22 that encodes for a purple fluorescent protein. The bacteria were added to a plasmid transformation tube and to a control tube; the tubes were heat shocked. After adding SOC media to both tubes, the tubes were incubated at 37 degrees and samples were transferred to agar plates to grow overnight. Ampillicin was added to a starter culture in LB supplemented tubes. After the cells had turned purple (Sample 1), the protein was harvested in an Allegra X-15 Centrifuge (Beckman Coulter, Inc, Brea, CA). The pellet was resuspended in 1x PBS solution with Lysozyme. Later, Bezonanse was added to the lysate, which was then centrifuged. Some of the supernatant was saved (Sample 2). The lysate was syringed filter to remove large particle matter. Finally, the protein was purified. First we added Ni-NTA buffer/resin mix to the protein. A Bio-Rad chromatography Econo column was prepared and the protein was isolated with a solution of 1x PBS with 250 mM Imidazole (Samples 2-6). A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the concentration of Sample 5. An electrophoresis module was assembled (Mini-PROTEAN tank) and filled with 1x TGS buffer. Samples were loaded in the following order, Standard, Flask, Soluble Fraction, Flow Through, Wash, Elution 1, Elution 2, Wash (*P), Elution 1 (*P), Elution 2 (*P). Electrophoresis produced a gel that was cleaned and dried. *P = Partner's samples.
Results:
Figure 1. Plate Images. Top Plate was Control Plate with no bacterial colonies. Bottom Plate was around 700 bacterial colonies transformed with pGEM-gbr22 plasmid. Fun Plate not shown.
Figure 2. Flask Image. Purple starter culture in LB media and Ampicillin after being incubated for approximately 24 hours.
Figure 3. Pellet remaining after centrifugation. Pellet weight was approximately 0.31 grams.
Figure 4. Elution 1 sample and Elution 2 sample after being eluted with 250 mM Imidazole in 1x PBS with Ni-NA resin/buffer.
Figure 5. Spectra Image for Elution 1 at 280 nm (Trial 1).
Figure 6. Spectra Image for Elution 1 at 280 nm (Trial 2).
Figure 7. Spectra Image for Elution 1 at 574 nm (Trial 1)
Figure 5. Spectra Image for Elution 1 at 574 nm (Trial 2).
Protein yields: 1.378 mg measured at both 574 nm and 280 nm.
Beer's Law Calculations
A = Ebc
Molecular weight = 25784 g/mol.
Yield at 280 nm
A = 0.435, E = 38850 M-1 cm-1, b = 1 cm.
c = A/Eb
= (1.12 x 10-5 M)*(25794.2 g/mol)
= 0.29 g/mol.
Yield = cV
= (0.29 g/mol)*(4.75 ml)
= 1.378 mg
Yield at 574 nm
A = 0.134, E = 118300 M-1 cm-1, b = 1 mm.
c = A/Eb
= (1.13 x 10-5 M)*(25794.2 g/mol)
= 0.29 g/mol
Yield = cV
= (0.29 g/mol)*(4.75 ml)
= 1.378 mg
Figure 8. Gel lanes before drying
Figure 9. Fermentas pre-stained molecular weight standard
Discussion:
At both 574 nm and 280 nm, the protein concentration was calculated to be 0.29 mg/mol. Consequently, the purified protein yield was 1.378 mg. We estimated the molecular weight of gbr22 to be approximately 28 kDa since the protein band in lane 7 was between the 26kDa band and 34kDa band of the standard. Our estimation is nearly 8% higher than the actual literature value. Since the other protein bands in lane 6 (Sample 5) are not as intense as the gbr22 band, we conclude that our protein is nearly 80% pure.
The purification process used several compounds to remove excess cellular baggage. Lysozyme broke down the cell walls of the bacteria while benzonanse removed nuclei acids from the protein solution. In this lab, we took six samples throughout the entire process to visualize what the compounds looked like after expression (Sample 1), purification (Samples 2-6). Sample 1 was obtained from the flask that contained bacterial cells in LB media and Ampicillin. Sample 2 was obtained after lysing the cell and removing large particle matter. Sample 3 contained the buffer/resin mix that was allowed to fall through the chromatography column. A wash step of 20 mM imidazole in 1x PBS was saved in Sample 4. Sample 5 contained the gbr22 protein after elution with a buffer of 250 mM imidazole in 1x PBS (Sample 5). Another 5 ml of the elution buffer was added and the fall out was saved in Sample 6. Since our protein contained a hexhistinide tag to the C-terminus, it bound to the Ni-NTA agarose. A small concentration of imidazole removed most of the other cellular proteins in the wash step, while a stronger concentration eluted the tagged protein in the elution steps. Most of the errors in this lab were probably systematic errors. For instance, sterile technique was not used at all times when working with bacterial cells due to negligence.
Conclusions:
In the present study, the coral protein gbr22 was expressed in E. Coli bacteria, purified by lysing the cells, removing insoluble debris and isolating with an affinity tag and Ni-NTA resin and finally characterized with SDS-PAGE analysis. We discovered that our final protein had a molecular weight of approximately 28 kDa and its purity was about 80%. The protocols in this study will be used again to study target proteins of interest for drug discovery.
References:
[1] Structural Genomics Consortium. Protein production and purification. Nat Methods. 2008;5:135–146. et al.
Understanding gbr-22 protein expression, purification and characterization
Introduction:
Proteins that are created from recombinant DNA from molecular cloning are known as recombinant proteins. These proteins are "used throughout biological and biomedical science" in areas such as x-ray crystallography and enzyme inhibition assays [1] . Some hosts for expressing the protein include bacteria, yeast, insect cells and human cells [1]. E. coli bacteria has become a popular expression host since many varieties of proteins can be over expressed in methods that "increase the yield of soluble protein" [1]. BL21 (DE3) is a common strain of E.coli that is used to quickly obtain large quantities of a protein. The plasmid that will be inserted into the expression host also contains some antibiotic-selection marker (e.g. ampicllin, carbenicillin) to identify bacterial colonies that successfully transformed with the plasmid [1]. After bacterial lysis through high-pressure homogenization or freeze-thaw procedures with lysozyme, the protein of interest is separated from extraneous proteins in a process known as protein purification [1]. To assist with protein purification, the modified recombinant protein includes an affinity tag, such as a C-terminal hexahistidine tag. The hexahistidine tag will bind to cations on a column matrix, and those proteins lacking the tag can be disposed of. Finally, the purified protein is characterized to ensure the protein is of adequate quality. SDS-PAGE analysis will denature the protein and show the quantity of the protein in proportion to the distance of migration in the gel. "This allows the purity of the sample to be estimated and whether the purified protein is of the expected size" [1]. UV-Vis spectroscopy is also used in this step to quantify the concentration of the protein. In this lab, the protein gbr22 was expressed, purified and characterized. As we complete the protocols in this lab, we expect the protein characterization to quantify the molecular weight of gbr22 near 25kDa, the literature value.
Materials & Methods:
Competent bacterial cells (New England BioLabs, E. Coli BL21(DE3)) were transformed with an expression plasmid pGEM-gbr22 that encodes for a purple fluorescent protein. The bacteria were added to a plasmid transformation tube and to a control tube; the tubes were heat shocked. After adding SOC media to both tubes, the tubes were incubated at 37 degrees and samples were transferred to agar plates to grow overnight. Ampillicin was added to a starter culture in LB supplemented tubes. After the cells had turned purple (Sample 1), the protein was harvested in an Allegra X-15 Centrifuge (Beckman Coulter, Inc, Brea, CA). The pellet was resuspended in 1x PBS solution with Lysozyme. Later, Bezonanse was added to the lysate, which was then centrifuged. Some of the supernatant was saved (Sample 2). The lysate was syringed filter to remove large particle matter. Finally, the protein was purified. First we added Ni-NTA buffer/resin mix to the protein. A Bio-Rad chromatography Econo column was prepared and the protein was isolated with a solution of 1x PBS with 250 mM Imidazole (Samples 2-6). A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the concentration of Sample 5. An electrophoresis module was assembled (Mini-PROTEAN tank) and filled with 1x TGS buffer. Samples were loaded in the following order, Standard, Flask, Soluble Fraction, Flow Through, Wash, Elution 1, Elution 2, Wash (*P), Elution 1 (*P), Elution 2 (*P). Electrophoresis produced a gel that was cleaned and dried. *P = Partner's samples.
Results:
Protein yields: 1.378 mg measured at both 574 nm and 280 nm.
Beer's Law Calculations
A = Ebc
Molecular weight = 25784 g/mol.
Yield at 280 nm
A = 0.435, E = 38850 M-1 cm-1, b = 1 cm.
c = A/Eb
= (1.12 x 10-5 M)*(25794.2 g/mol)
= 0.29 g/mol.
Yield = cV
= (0.29 g/mol)*(4.75 ml)
= 1.378 mg
Yield at 574 nm
A = 0.134, E = 118300 M-1 cm-1, b = 1 mm.c = A/Eb
= (1.13 x 10-5 M)*(25794.2 g/mol)
= 0.29 g/mol
Yield = cV
= (0.29 g/mol)*(4.75 ml)
= 1.378 mg
Discussion:
At both 574 nm and 280 nm, the protein concentration was calculated to be 0.29 mg/mol. Consequently, the purified protein yield was 1.378 mg. We estimated the molecular weight of gbr22 to be approximately 28 kDa since the protein band in lane 7 was between the 26kDa band and 34kDa band of the standard. Our estimation is nearly 8% higher than the actual literature value. Since the other protein bands in lane 6 (Sample 5) are not as intense as the gbr22 band, we conclude that our protein is nearly 80% pure.
The purification process used several compounds to remove excess cellular baggage. Lysozyme broke down the cell walls of the bacteria while benzonanse removed nuclei acids from the protein solution. In this lab, we took six samples throughout the entire process to visualize what the compounds looked like after expression (Sample 1), purification (Samples 2-6). Sample 1 was obtained from the flask that contained bacterial cells in LB media and Ampicillin. Sample 2 was obtained after lysing the cell and removing large particle matter. Sample 3 contained the buffer/resin mix that was allowed to fall through the chromatography column. A wash step of 20 mM imidazole in 1x PBS was saved in Sample 4. Sample 5 contained the gbr22 protein after elution with a buffer of 250 mM imidazole in 1x PBS (Sample 5). Another 5 ml of the elution buffer was added and the fall out was saved in Sample 6. Since our protein contained a hexhistinide tag to the C-terminus, it bound to the Ni-NTA agarose. A small concentration of imidazole removed most of the other cellular proteins in the wash step, while a stronger concentration eluted the tagged protein in the elution steps. Most of the errors in this lab were probably systematic errors. For instance, sterile technique was not used at all times when working with bacterial cells due to negligence.
Conclusions:
In the present study, the coral protein gbr22 was expressed in E. Coli bacteria, purified by lysing the cells, removing insoluble debris and isolating with an affinity tag and Ni-NTA resin and finally characterized with SDS-PAGE analysis. We discovered that our final protein had a molecular weight of approximately 28 kDa and its purity was about 80%. The protocols in this study will be used again to study target proteins of interest for drug discovery.
References:
[1] Structural Genomics Consortium. Protein production and purification. Nat Methods. 2008;5:135–146. et al.
[2] Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/index.html(accessed April 16).